The selection of one or more specific cell phenotypes from a heterogeneous cell composition, e.g. blood or bone marrow, has particular utility for cellular and gene therapies. For example, it has been demonstrated that the selection of cells expressing the CD34 antigen has utility in several therapies, such as a part of an adjunctive treatment for cancer (Civin, U.S. Pat. Nos.: 5,035,994; 4,965,204; 5,081,030; 5,130,144). The selection of specific target cells for genetic manipulation is also of particular interest.
There are numerous cell selection techniques. For example, quiescent CD34+ cells may be selected by treating a hematopoietic cell culture with a chemical such as 5-fluorouracil which selectively kills dividing cells (Berardi, A. C. et al., Science 267:104-108, 1995). One particularly useful approach utilizes the selective binding of antibodies. Antibodies naturally bind to a specific antigen expressed by only certain cells. By matching an antibody to a specific cellular antigen, such cells may be physically removed or identified in a heterogeneous cell population. For discussions of antibody selection see Areman, E. et al., Eds. Bone Marrow and Stem Cell Processing, F. A. Davis Company, Philadelphia, 1992, and Gee, A. P., et al, Eds. Advances in Bone Marrow Purging and Processing, Wiley-Liss, New York, 1993.
Cellular selection techniques generally fall with two broad categories, negative cell selection and positive cell selection. As the terms imply, negative selection involves the removal of selected cell phenotypes from a population, while positive selection involves the selection or isolation of a specific cell phenotype from a larger heterogeneous cell population.
Negative cell selection techniques have found use in the removal of potentially harmful cells from a patient's or a donor's blood or bone marrow. For instance, a treatment for metastatic cancer may involve removal of a sample of the patient's bone marrow prior to ablative chemotherapy or radiation, with the intent to replace the patient's bone marrow cells after the ablative therapy in order to replenish hematopoietic cells. To minimize the risk of returning metastatic tumor cells to the patient, negative cell selection or purging is applied to the patient's bone marrow sample prior to reinfusion. One method of performing this negative cell selection involves the use of anti-tumor antibodies linked to a solid phase, such as magnetic beads, for binding the tumor cells and removing from blood, see (Hardwick, A., et al., J Hematotherapy 1:379-386, 1992). Negative selection of cells using lysis or enzymatic elimination of certain cells has also been employed (Areman, et al., supra).
As stated, positive selection involves targeting and separating a specific cell phenotype from a heterogeneous cell population. For example, cells expressing the CD34 antigen have been selected for use in bone marrow transplantation (Gee, et al., supra). While selection techniques employing toxic agents, e.g., (lytic agents), have been employed to eliminate certain cell types, the selectivity of such approaches are limited to removal or elimination of certain cells, not the affirmative selection of a specific cell type.
The use of antibodies for binding to specific cells has found widespread utility in positive selection techniques (Gee, et al., supra). One approach involves tagging or binding to the antibody a fluorescent dye and passing the antibody bound to the cell through a sorter. The cells to which the antibodies bind are identified and segregated by fluorescence-activated cell sorting (FACS). Another technique involves the binding of the antibody to a solid phase support or particle. Passing a cell composition past the antibody bearing support allows the antibodies to bind and hold the desired cells, thus removing the desired cells from the composition. Incubating a cell composition with antibody bearing particle, i.e., paramagnetic particles, allows for the separation of the particle bound cells from the remainder of the population, i.e., through magnetic separation (Gee et al., supra, pp.293-302).
The captured cells must be released from any solid support after the selection process, but in such a manner so as to maintain viability of the captured cells. Further, some researchers maintain that continued binding of an antibody or antibody fragment to the cell effects the usefulness of the cell (Berardi, et al. supra).
A particular concern with any positive cell selection technique employing an antibody based mechanism, is the retention of viability of the desired cells while effecting their release from the antibody and solid phase separation material. Release of the cells through variation of the surrounding pH and temperature is difficult since the pH must be maintained at around 7.0-7.4, and the temperature cannot be raised much higher than 37.degree. C.
Certain cell types may tolerate low levels of reducing agents such as dithiothreitol and/or chelating agents such as EDTA, while other target cells may not remain viable even under very mild reducing or chelating conditions.
The strong affinity of avidin for biotin has been employed to effect the binding of cells to antibody bearing solid supports.
In avidin/biotin based techniques, typically an antibody which is specific for the target cell is biotinylated according to one of several standard methods (Avidin-Biotin Chemistry: A Handbook, Eds. Savage, M. D., et al., Pierce Chemical Co, 1992). For negative selection, the target cell is bound by the biotinylated antibody, which in turn is bound to an avidin-coated solid phase, usually in column form. The non-bound cells are then recovered, and the negatively selected cells bound to avidin are discarded.
For positive cell selection, however, the very strong affinity of avidin for biotin is disadvantageous since the target cells are firmly held within the cell/antibody-biotin/avidin complex. Since the avidin/biotin interaction is so strong, the disruption of other bonds was proposed for the release of desired target antigens. Certain biotinylating agents have chemically cleavable covalent bonds within their spacer arms or form cleavable covalent bonds with target proteins (Sigler, G. F. U.S. Pat. Nos.: 4,798,795 and 4,709,037; Wilchek, M., et al, German Pat. App. DE 3629194 A; Avidin-Biotin Chemistry: A Handbook, supra, p.41). The bonds are cleaved under reducing conditions employing dithiothreitol, mercaptoethanol, or sodium borohydride, but these conditions are generally too damaging to cells to be considered for selection of cells which must remain functional.
Other techniques involve the competitive displacement of biotin from the avidin support, leaving the biotinylated antibody bound to the cell. Alternatively, a biotin-analog is covalently bound to a primary antibody which binds to the cell of interest. The cell/antibody/biotin-analog complex is bound by a secondary anti-biotin antibody, bound to a solid support, for separation from the heterogeneous cell mixture. Then the cell/antibody/biotin-analog complex is released from the secondary antibody by competition with biotin. This method necessarily leaves the antibody bound to the cell (Al-Abdaly, F. et al., WO 95/07466).
Several techniques for positive cell selection rely on mechanical means for disruption of antibody/epitope interactions for release of selected cells. Tissue culture flasks may be coated with a primary antibody which binds the target cells; after the unbound cells are washed away, the target cells are released by striking the sides of the flask (Lebkowski, J. S., et al., Transplantation 53:1101-1019, 1992). Another method for positive cell selection employs a "sandwich" technique which involves avidin bound to a biotinylated secondary antibody which binds a primary antibody, which in turn binds the target cell to form a complex. After separation of the complex from the heterogeneous cell suspension, the target cell is removed from the avidin by agitation to disrupt the interaction between the secondary and primary antibodies (Berenson, R. J., et al., U.S. Pat. Nos.: 5,215,927 and 5,225,353). Mechanical release is disadvantageous for the obvious reason that cells may sustain damage during the release process, and it has been reported that low numbers of viable cells are recovered after mechanical release (Egeland, T., et al., Scand J. Immunol. 27: 439-444, 1988). There is also the possibility that antibody fragments might adhere to the cells.
Another method for cell release involves proteolysis by enzymes such as papain and chymopapain. The target cells may be bound to magnetic beads via a primary antibody which is in turn bound to magnetic beads. After the cell/antibody/bead complex is removed from the heterogeneous cell suspension, the cells are released from the beads by proteolysis of the cell surface antigen or the antibody, or both (Hardwick, A., et al., J. Hematotherapy 1:379-386, 1992; Civin, C. E., et al., In Bone Marrow purging and Processing Progress in Clinical And Biological Research, Vol. 333, Eds. S. Gross, et al., Alan R. Liss, Inc, New York, pp 387-402; Civin, C. I., EP 0 395 355 A1; Hardwick, A., et al., In Advances in Bone Marrow Purging and Processing-Progress in Clinical and Biological Research, Vol. 377, Eds. Worthington-White, D. A., et al., Wiley-Liss, Inc., New York, pp 583-589). Proteolysis by papain or chymopapain is advantageous over mechanical disruption because these enzymes are not generally harmful to cells. However, enzymes digest cell surface proteins which could be important for the proliferation, differentiation, and homing of hematopoietic stem cells, for instance. Moreover, the digestion of cell surface proteins makes subsequent negative selection difficult or impossible.
Another technique involves the competitive displacement of the antibody from the cell antigen using additional antibody or antibody fragments. However, while this approach effects the release of a cell from a solid support, at least a portion of an antibody remains bound to the resulting cell, which may be detrimental (Berardi, et al., supra).
There remains a need for a positive cell selection method which produces a high yield of functional target cells, and which relies on relatively inexpensive, benign reagents in a physiologically compatible solution. Moreover, there remains a need for a positive cell selection method which leaves cell surface proteins intact. It would also be advantageous to have a method which leaves the positively selected cells free from antibodies or other ligands bound to the cell surface.